Immunotherapy with antigen-expressing dendritic cells (DCs) has been proposed as an approach for immunotherapy of viral infections and cancer. However, current methods of loading DCs with tumor associated antigens, ranging from transduction with viral vectors to loading DCs with peptides, proteins or nucleic acids are either not safe for application in humans or fail to elicit a satisfying anti-tumor effect. In a recent study, we used the cross-priming pathway to induce a protective T cell response against hepatitis C virus (Immunity, 2004;20:47-5). For this purpose, vaccine DCs were transfected with self-replicating viral RNAs (RNA replicons) of bovine viral diarrhea virus (BVDV), in which the genetic units coding for the virus structural proteins were replaced by a heterologous open reading frame coding for an antigen of choice. Such bi-cistronic BVDV replicons turned out to be advantageous for vaccination because they express high amounts of the antigen in the cytoplasm of the transfected cells and do not form infectious virus particles. An additional advantage is the cytopathogenicity of BVDV replicons (DC/cpBVDV) that express the viral NS3 protein resulting in apoptosis 24-48 h after transfection. This time-delayed apoptosis of the replicon-transfected vaccine DCs was shown to be crucial for efficient cross-priming of an HCV antigen-specific T cell response. The aim of the current study was to employ the DC/cpBVDV replicon system to vaccinate against neoplastic cells that over-express a model tumor antigen. Using the breast cancer antigen Her2 as a model antigen, we constructed two DC/cpBVDV replicons encoding its extracellular domain and its middle fragment, respectively. Mice were immunized twice subcutaneously with Her2-encoding replicon-transfected dendritic cells and subsequently challenged with Her2-expressing breast cancer cells. Tumor growth was measured thrice weekly for a period of 3 weeks. Immunization with replicon-loaded DCs resulted in significantly smaller tumors compared to mock vaccination (p<0.02). Furthermore, 33% and 50% of the mice remained tumor-free after immunization with the respective replicon vaccines, whereas all mock-immunized mice developed tumors. We further demonstrated that protection against tumors correlated with Her2-specific T cell responses and that antibody responses were not induced. Furthermore, depletion of CD4+ or CD8+ cells prior to tumor challenge abrogated the anti-tumor effect. Her2-specific CD8 T cells were induced by cross-priming, because they produced IFN-gamma in response to H-2q-positive antigen-presenting cells in vitro, even though the transfected DCs used for immunization expressed H-2b. In conclusion, this study shows that cytopathic RNA replicons expressing a tumor antigen mediate a significant anti-tumor effect by the induction of T cell cross-priming. Our approach may be useful as a safe and efficient means to load DCs with a tumor antigen in cancer immunotherapy.